Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase

Article abstract of DOI:10.1016/j.bmcl.2018.01.058

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ～80% at 2 h when dosed in mice orally at 50 mg/kg.

Full text of DOI:10.1016/j.bmcl.2018.01.058

Accepted Manuscript
Novel Nicotinamide Analog as Inhibitor of Nicotinamide N-methyltransferase
Sven Ruf, Mahanandeesha Siddappa Hallur, Nisha K Anchan, Indu N Swamy,
Karthikai Raj Murugesan, Sayantani Sarkar, Lokesh Kananti Narasimhulu, V.P.
Rama Kishore Putta, Shama Shaik, Devaraj Venkatapura Chandrasekar, Vishal
Subhash Mane, Sanjay Venkatachalapathi Kadnur, Juluri Suresh, Ravi Kanth
Bhamidipati, Manvi Singh, Raghunadha Reddy Burri, Rajendra Kristam,
Herman Schreuder, Joerg Czech, Christine Rudolph, Alexander Marker,
Thomas Langer, Ramesh Mullangi, Takeshi Yura, Ramachandraiah Gosu, Aimo
Kannt, Saravanakumar Dhakshinamoorthy, Sridharan Rajagopal
PII:
S0960-894X(18)30069-6
https://doi.org/10.1016/j.bmcl.2018.01.058
BMCL 25584
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2017
25 January 2018
27 January 2018
Please cite this article as: Ruf, S., Hallur, M.S., Anchan, N.K., Swamy, I.N., Murugesan, K.R., Sarkar, S.,
Narasimhulu, L.K., Putta, P.R.K., Shaik, S., Chandrasekar, D.V., Mane, V.S., Kadnur, S.V., Suresh, J., Bhamidipati,
R.K., Singh, M., Burri, R.R., Kristam, R., Schreuder, H., Czech, J., Rudolph, C., Marker, A., Langer, T., Mullangi,
R., Yura, T., Gosu, R., Kannt, A., Dhakshinamoorthy, S., Rajagopal, S., Novel Nicotinamide Analog as Inhibitor
of Nicotinamide N-methyltransferase, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/
10.1016/j.bmcl.2018.01.058
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Novel Nicotinamide Analog as Inhibitor of
Nicotinamide N-methyltransferase
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Sven Rufa*, Mahanandeesha Siddappa Hallurb, Nisha K Anchanb, Indu N Swamyb, Karthikai Raj Murugesanb ,
Sayantani Sarkarb, Lokesh Kananti Narasimhulub, ,V P Rama Kishore Puttab, Shama Shaikc,, Devaraj
Venkatapura Chandrasekard, Vishal Subhash Manec, Sanjay Venkatachalapathi Kadnurc, Juluri Sureshc, Ravi
Kanth Bhamidipatid, Manvi Singhe, Raghunadha Reddy Burrie, Rajendra Kristame, Herman Schreudera. Joerg
Czecha, Christine Rudolpha Alexander Markerf, Thomas Langerg, Ramesh Mullangid, Takeshi Yurab,
Ramachandraiah Gosud, Aimo Kannth, Saravanakumar Dhakshinamoorthyc and Sridharan Rajagopalb*
Abstract: Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have
identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and
reduced the formation of 1-methyl- nicotinamide (MNA), the primary metabolite of NA by ~80% at 2h when
dosed in mice orally at 50mg/kg.
Compound 12 Human/ mouse NNMT enzymatic inhibition (IC50): 0.58/ 4.5 µM
Inhibition of MNA in U2OS/ 3T3L1 cell based assay: 0.3/2.1 µM
Human/ mouse metabolic stability (% remaining after 30m): 85/ 69
In-vivo target engagement (MNA reduction, 2h): ~80%

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Novel Nicotinamide Analog as Inhibitor of
Nicotinamide N-methyltransferase
Sven Rufa*, Mahanandeesha Siddappa Hallurb, Nisha K Anchanb, Indu N Swamyb, Karthikai Raj Murugesanb ,
Sayantani Sarkarb, Lokesh Kananti Narasimhulub, ,V P Rama Kishore Puttab, Shama Shaikc,, Devaraj Venkatapura
Chandrasekard, Vishal Subhash Manec, Sanjay Venkatachalapathi Kadnurc, Juluri Sureshc, Ravi Kanth Bhamidipatid,
Manvi Singhe, Raghunadha Reddy Burrie, Rajendra Kristame, Herman Schreudera. Joerg Czecha, Christine Rudolpha
Alexander Markerf, Thomas Langerg, Ramesh Mullangid, Takeshi Yurab, Ramachandraiah Gosud, Aimo Kannth,
Saravanakumar Dhakshinamoorthyc and Sridharan Rajagopalb*
aIntegrated Drug Discovery, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany
bDepartment of Medicinal Chemistry, Jubilant Biosys Ltd, Bangalore-560022, India
cDepartment of Discovery Biology, Jubilant Biosys Ltd, Bangalore-560022, India
cDepartment of Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Bangalore-560022, India
dDepartment of Structural Biology, Jubilant Biosys Ltd, Bangalore-560022, India
eDepartment of Computational Chemistry, Jubilant Biosys Ltd, Bangalore-560022, India
fR&D Drug Metabolism & Pharmacokinetics, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany
gBiologics Research, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany
hR&D Diabetes, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany & Institute of Experimental
Pharmacology, Medical Faculty Mannheim, University of Heidelberg, D-68167 Mannheim, Germany
* corresponding authors: Tel: +49 69 305 12982/+91 80 66628364
Email Address: Sven.Ruf@sanofi.com/sridharan_rajagopal@jubilantbiosys.com

ARTICLE INFO
ABSTRACT
Abstract: Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes.
We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT
enzymatic activity and reduced the formation of 1-methyl- nicotinamide (MNA), the primary
metabolite of NA by ~80% at 2h when dosed in mice orally at 50mg/kg.
Article history:
Received
Revised
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Nicotinamide N-methyltransferase (NNMT), 1-
incotinamide, Methyl-nicotinamide (MNA),
Obesity, diabetes,
Obesity is considered as one of the major risk factors
for development of insulin resistance and Type 2 diabetes (T2D).
Nicotinamide N-methyl transferase (NNMT), an enzyme that
catalyzes the transfer of methyl group from S-adenosyl-L-
methionine (SAM) to nicotinamide (NA) to form N-
methylnicotinamide (MNA) is implicated in the regulation of
body weight and insulin sensitivity. NNMT is expressed in the
liver, adipose and other tissues1,2. Several reports have suggested
the role of NNMT in various disease conditions including
metabolic disorders3-5. Increased expression of NNMT has also
been linked to enhanced cell proliferation and disease
progression in a wide variety of cancers. It is also known that
NNMT is up-regulated in Parkinson’s disease6,,7. Recent reports
have highlighted the relationship between the high expression of
We undertook an effort to identify small molecule
inhibitors of NNMT that could be used to understand the
mechanism of NNMT inhibition and can further be developed as
drug candidates to treat metabolic diseases. Here, for the first
time, we report a small molecule inhibitor of NNMT that
modulates MNA levels in an in-vivo target engagement study.
In order to find novel small molecule inhibitors of
NNMT, we undertook a high throughput screening (HTS)
campaign of an in-house library of compounds18. Several hits
were identified including compound 1 that was found to be active
in human and mouse NNMT enzymatic and cell based assays19.
To understand the SAR around compound 1, a focused library of
compounds was synthesized as shown in Table 1. Replacement
of the amide moiety with other groups such as a reverse amide
(3), tetrazole (4),-CONHEt (5), and amidine (6), resulted in loss
of activity. Replacement of the –OMe group with –OCD3 (2) led
to improvement of ~2 fold in activity (Table 1).
NNMT and obesity/T2D8-12
.
NNMT expression is increased in adipocytes of obese
compared to non-obese PIMA Indians13 Knockdown of NNMT
expression using an antisense oligonucleotide has been reported
to reduce fat mass, body weight and increase the energy
expenditure11. Population studies indicate that MNA levels
strongly correlate with obesity and diabetes in Chinese
population14. MNA levels were found to be elevated in the urine
samples of humans with T2D, and in diabetic animals (db/db,
obese Zucker rats). In addition, a recent patent disclosure
suggests MNA as a soluble biomarker for insulin resistance in
patients15.
Table 1: SAR around compound 1,
hNNMT
Compound
Code
Structure
enzymatic
assay
(IC50,nM)
JBSNF-
000088
1
2
3
2445±79
1268±346
>30000
O
JBSNF-
000292
NH2
D
D
O
N
D
Recently, quaternary methyl salts of quinoliniums were
published as potent inhibitors of NNMT16. We have recently
published the crystal structure of MNA in a bound form with the
active site of NNMT enzyme and for the first time demonstrated
that MNA can bind to the active site of NNMT, thereby inhibit
the NA binding17.
JBSNF-
000156
N
N
N
JBSNF-
000198
N
4
>30000
H
O
N

O
rings of NCA and compound 12. B. The ligand interaction
diagram of compound 12 schematically showing the key
interactions. Magenta-colored arrows show H-bond interactions,
while green-colored arrow shows aromatic interactions.
N
JBSNF-
000199
5
6
>30000
>30000
H
O
N
NH
JBSNF-
000408
NH2
Compound 12 binds in a similar way as nicotinamide (NA). The
amide carbonyl picks up a H-bond interaction with the side-chain
of Ser-213, the NH2 of the amide picks an H-bond interaction
with Leu-164 backbone carbonyl and the secondary amine
(NHCH3) on the 3- position of the pyridine ring picks an H-bond
interaction with the hydroxyl side-chain of Tyr-20. The pyridine
ring also shows π-stacking with the phenyl ring of Tyr-204
similar to that in NCA. The amide moiety of compound 12 can
also bind in a flipped orientation and pick an H-bond interaction
with Ser-201.
O
N
We also explored substitution of the pyridine ring. Replacement
of the 6-methoxy group of compound 1 with methyl (7),
trifluoromethyl (8), ethyl (9), -SMe (10), and OCHF2 (11)
substituents led to compounds that were either inactive or less
potent. The difference in potency could be due to both steric and
electrostatic effects. When the methoxy group was replaced with
–NHMe (12), there was 3 fold increases in activity. Replacing –
NHMe with the secondary amine -N(Me)2 (13) led to complete
loss of activity. It is clear from the above SAR that the –NH is
important for the activity. We carried out a molecular docking
study to understand the nature of interactions between compound
12 and the active site residues of hNNMT using the Glide
module of Schrodinger 2016-2 release20. The publicly available
PDB structure, 3ROD (chain A), was used for this docking
analysis. The predicted binding mode of compound 12 and its
key interactions are shown in Figure 1.
The replacement of 6-methoxy group of compound 1 with
various groups (except -OCD3 and -NHCH3) led to a loss of
potency. This could be due to steric clashes of these substituted
groups with the nearby residues. One such example is compound
8 that shows its CF3 group clashing with Tyr-20 and Tyr-24 as
shown in Figure 2.
Figure 1: A. Predicted binding mode of compound 12 (magenta
colored) in hNNMT active site (PDB: 3ROD.pdb). Green lines
indicate the H-bond interactions between the NCA (green
colored) and the protein active site residues, whereas magenta
lines indicate the H-bond interactions between compound 12 and
the protein active site residues. Black line indicates the aromatic
(π-stacking) interactions between Tyr-204 with both the pyridine
Figure 2: Steric clashes of -CF3 of compound 8 with Tyr-20
and Tyr-24 of hNNMT (PDB: 3ROD.pdb)
Substitution of pyridine at other positions was explored to further
understand the SAR of compound 1. Compound 15 with methyl

O
substitution at the 4- position led to 3 fold increase in activity,
whereas substitution at other positions such as 4-OEt (14), 2-OEt
(16), 5-F (17), 2-Me (18), led to complete loss of activity.
Replacing the pyridine with other ring system such as pyridone
(19), pyrazine (20), phenyl (21), and pyridazine (22) led to loss in
potency (Table 2).
22
JBSNF-
000516
>10000
NH2
N
N
O
Based on the SAR studies, compound 12 was selected
for further biological evaluation15,19,21 and the data is shown in
Table 3. Compound 12 inhibited the NNMT enzymatic activity in
in-vitro biochemical as well as the cell based assay. To evaluate
the in-vivo profile of compound 12, we first assessed the
pharmacokinetics of compound 12 (Table 3). Furthermore, the in
vivo target engagement study was conducted using C57Bl6/N
mice21. The mice were dosed orally with 50mg/kg of compound
12 and the MNA levels were measured using LC-MS/MS for
various time periods up to 24h (Figure 3). We observed a
decrease in MNA levels for over 2h post dosing of compound 12
(Figure 3). This clearly suggests that compound 12 is able to
engage the NNMT target to achieve enzymatic inhibition
resulting in MNA reduction22.
Table 2: SAR of compound 1
hNNMT
Compound
Code
Structure
enzymatic
assay
(IC50, nM)
>10000
O
JBSNF-
000172
NH2
7
H3C
N
O
O
>30000
NH2
JBSNF-
000174
F
F
8
9
N
F
JBSNF-
000176
>30000
>30000
NH2
Table 3: Profile of compound 12
N
10
11
JBSNF-
000418
Compound 12
O
JBSNF-
000419
>30000
NH2
O
N
m.wt(Da)/clogP/tPSA(Å2)
151/0.3/67.5
F
F
O
12
13
14
JBSNF-
000265
588±75
>30000
>30000
NH2
human/mouse NNMT assay
0.58/4.5
IC50 (µM)
N
N
N
H
O
JBSNF-
000343
NH2
U2OS/3T3L-1 cell based assay
N
0.3/2.1
IC50 (µM)
JBSNF-
000238
O
O
NH2
N
Metabolic stability h/r/m (%
85/97/69
O
15
16
JBSNF-
000581
837±71
>30000
NH2
remaining at 30min)
O
N
O
O
JBSNF-
000126
NH2
Solubility (µM)
~200
1.2
O
N
O
17
JBSNF-
000212
>30000
Caco2 Efflux ratio
F
NH2
O
N
N
O
18
19
JBSNF-
000116
>30000
>30000
Cyp Inhibition (3A4, 2D6, 2C9,
2C19)IC50 (µM)
NH2
>20
O
O
JBSNF-
000115
NH2
O
N
Cmax PO/IV (10/1 mg/kg)
AUC PO/IV (ng.h/mL)
t1/2 PO/IV (h)
1433/587
1087/511
1.2/0.8
O
20
21
JBSNF-
000314
N
N
>30000
>30000
NH2
O
O
JBSNF-
000111
NH2
O

nicotinamide-N-methyltransferase as a novel serum tumor marker
for colorectal cancer. Clin. Cancer Res. 2005, 11 (18), 6550−6557.
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Cl/Vd/F%
27.9/1.85/19
mL/min/kg and L/Kg
MNA reduction upon dosing of
~80% reduction
for 2h
50mg/kg
compound
p.o
(Target engagement)
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Figure 3: In vivo target engagement of compound 12
In summary, we report a novel nicotinamide analog (compound
12) as a potent small molecule inhibitor of NNMT and for the
first time demonstrate that a small molecule inhibitor of NNMT
could be used to modulate NNMT activity in-vivo. Efficacy
studies using DIO mice model is planned to evaluate the efficacy
of compound 12.
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BSA, 2mM Dithiothreitol and 1% DMSO. The fluorescent product
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et al, 2016. In brief, 3 µl of human NNMT (Emelca Bioscience)
were added to 3 µl of test compound (30 µM) in a black 384-well
small volume microtiter plate. After incubation for 30 minutes at
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of 3 µl substrate solution containing 60 µM nicotinamide and 60
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µg/ml NNMT, 10 µM test compound, 20 µM nicotinamide and 20
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21) Male C57Bl6/N mice (Vivo Bio Tech Ltd, India) were used for
the target engagement study. Animals were dosed with compound
12, 50 mg kg-1, p.o. and at every time point 3 animals were bled
and sacrificed. Animals were bled and sacrificed after 0.25, 0.5, 1,
2, 4, 6, 8 and 24h of dosing. 3 animals were bled and sacrificed at
0 min (no treatment). Plasma was harvested by centrifuging the
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achieved by MS/MS detection in positive ion mode for MNA and
internal standard (d4–MNA). Detection of the ions was performed
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activity was carried out using fluorescence method. MNA formed
in the NNMT reaction reacts with acetophenone in the presence of
KOH and formic acid and forms a fluorescent product, 2, 7-
naphthyridine which was measured. The final assay reaction
mixture contained a buffer of 100mM Tris Hcl pH 7.5, 0.04%
22) All synthetic procedures and IC50 curves of selected compounds
are given in the supplementary information

In-vivo target engagement (MNA reduction, 2h):
~80%
Graphical Abstract
Novel Nicotinamide Analog as Inhibitor of
Nicotinamide N-methyltransferase
Sven Rufa*, Mahanandeesha Siddappa Hallurb, Nisha K
Anchanb, Indu N Swamyb, Karthikai Raj Murugesanb ,
Sayantani Sarkarb, Lokesh Kananti Narasimhulub, ,V P Rama
Kishore Puttab, Shama Shaikc, Devaraj Venkatapura
Chandrasekard, Vishal Subhash Manec, Sanjay
Venkatachalapathi Kadnurc, Juluri Sureshc, Ravi Kanth
Bhamidipatid, Manvi Singhe, Raghunadha Reddy Burrie,
Rajendra Kristame, Herman Schreudera, Joerg Czecha,
Christine Rudolpha, Alexander Markerf, Thomas Langerg,
Ramesh Mullangid, Takeshi Yurab, Ramachandraiah Gosud,
Aimo Kannth, Saravanakumar Dhakshinamoorthyc and
Sridharan Rajagopalb*
Abstract: Nicotinamide N-methyltransferase (NNMT)
has been linked to obesity and diabetes. We have
identified a novel nicotinamide (NA) analog, compound
12 that inhibited NNMT enzymatic activity and reduced
the formation of 1-methyl- nicotinamide (MNA), the
primary metabolite of NA by ~80% at 2h when dosed in
mice orally at 50mg/kg.
Compound 12 Human/ mouse NNMT enzymatic
inhibition (IC50): 0.58/ 4.5 µM
Inhibition of MNA in U2OS/ 3T3L1 cell based
assay: 0.3/2.1 µM
Human/ mouse metabolic stability (% remaining
after 30m): 85/ 69